GnRH-1 (also known as LHRH) neurons, critical for reproduction, are derived from the nasal placode and migrate into the brain where they become integral members of the hypothalamic-pituitary-gonadal axis. We study mechanism(s) underlying GnRH-1 neuronal differentiation, migration and axonal targeting in normal/transgenic animals, and nasal explants. Using these same models, our work also addresses the mechanisms regulating (intrinsic and trans-synaptic) GnRH gene expression, peptide synthesis and secretion in GnRH-1 neurons. Multiple approaches are used to identify and understand the multitude of molecules and factors which play a role in directing the GnRH-1 neurons to their final location in the CNS. These include differential screening of libraries obtained from migrating versus non-migrating cells, examination of molecules differentially expressed at key locations along the migratory route, morphological examination of the development of the GnRH-1 system in knockout mice, and perturbation of molecules in vitro and subsequent monitoring of GnRH-1 neuronal movement. As GnRH-1 neurons migrate they also mature and the two processes may in fact be linked. To investigate the maturation of GnRH-1 neurons we use calcium imaging, electrophysiology and biochemical measures to examine GnRH-1 neuronal activity and peptide secretion. [unreadable] Over the past year we have investigated the role of the brain-gut peptide, CCK, in GnRH-1 neuronal development, both as a factor regulating neuronal migration and well as GnRH-1 neuronal activity. Functional analysis indicated that CCK inhibits GnRH neuronal movement and after migration has ceased, down-regulates GnRH-1 neuronal activity. In addition voltage-gated calcium channels were examined in the developing GnRH-1 system. We have continued studies on the influence of GABA on GnRH-1 neuronal migration and evaluated the GABAA receptor subunits expressed in GnRH-1 cells as a function of development. These studies indicated that the N-type VGCC modulated GnRh-1 neuronal migration. Studies have been performed to characterize, and identify the role of GnRH-1 in developing incisor. From these studies, morphological changes in the incisor of GnRH-1 mutant mice have been identified. These studies also show that the GnRH receptor is expressed in the developing tooth, providing a signal transduction pathway for GnRH-1 to act. Collaborative studies have focused on the role of GnRH-1 itself as an autocrine or paracrine regulator of GnRH-1 neuronal activity. The expression of the GnRH-1 receptor was verified on GnRH-1 cells and inhibition of GnRH-1 shown to alter GnRH-1 neuronal activity in a concentration dependent manner. A second collaborative study examined the development of the GnRH systems in zebrafish to determine whether GnRH-1 cells arise in association with the nasal placode in vertebrates. Using morphological studies as well as tagged GnRH-1 cells in live embryos, GnRH-1 cells were observed migration from the nasal placode region into the developing forebrain. Studies in progress center on the role of NELF (a ?migrational? molecule), cytokines, and growth factors in GnRH-1 development as well as in situ characterization of the migration of LHRH neurons (real time microscopy). We have recently obtained our first NELF KO animals. These mice are fertile and will be used to examine changes, if any, in the absence of NELF. In addition, we continue to study the role of estrogen on GnRH-1 neuronal activity and have recently start monitoring GnRH-1 neuronal activity in nasal explants generated from estrogen receptor knockout mice. Studies in progress examine the electrical properties associated with GnRH-1 neuronal activity (combining electrical recording and calcium imaging). Future studies are designed to expand upon our present results and are directed at the molecules and cues important for development of the olfactory and GnRH-1 neuronal systems as well as the mechanisms regulating GnRH-1 neuronal activity. Specific studies in progress focus on: 1) isolation of midline cues which influence olfactory axon outgrowth; 2) the role of NELF and other molecules in GnRH-1 migration, 3) identifying pacemaker molecules in GnRH-1 neurons that participate in establishment/maintenance of rhythmic activity as well as regulator molecules such as Kisspeptin, 4) GABAergic signals during development of the GnRH-1 system and 5) the mechanisms by which estrogen alters GnRH-1 neuronal activity.